[Development of multiplex reverse translation-polymerase chain reaction methods for detection of dengue virus type 1-4 and its application in clinical use].

OBJECTIVE

To develop multiplex reverse translation-polymerase chain reaction (RT-PCR) method for detection of dengue virus type 1-4.

METHODS

Based on the genomes sequence analysis of dengue virus type 1-4, four-pair of primers were designed. The specificity of the primers was primarily tested by searching the GenBank DNA sequence database. The optimal reaction conditions of the multiplex RT-PCR were then established. The specificity of RT-PCR was tested using the homologous yellow fever virus and Japanese encephalitis virus. 30 serum samples of dengue virus from suspected sufferers in the prevalence of dengue virus in 2003 were detected using the methods we developed.

RESULTS

Positive segments about 295, 237, 118, 347 bp could be seen in the multiplex RT-PCR production of dengue virus type 1-4, respectively. There were no positive segments in the RT-PCR productions of Japanese encephalitis virus and yellow fever virus. 25 of the 30 serum samples showed dengue virus type 1 positive results, while the sequencing results suggesting the amplification sequence having a high homology with dengue virus type 1 strain Cambodia, GD14/97 and GD05/99 (97%, 97%, 98%, respective).

CONCLUSION

The method of multiplex RT-PCR we established could be used for early detection and identification of dengue virus type 1-4.